I wish to include a fatty acid treatment in my endothelial cell culture. Please suggest what mixtures would be suitable from the point of activation.
Yes Manindra. You have to prepare a solution of bovine free fatty acid and add the fatty acid you want to incorpórate, the ratio of fatty acid per albumin is maximun 10:1, oleic acid, docosahexanoic acid or eicosapentanoic acid are easy to handle in the salt form. The solution of albumin 32 mg/ml should be in PBS 7.4 and calculate, based on the previous ratio the amount of fatty acid you have to add, mix at 37 degrees. Alternatively if you have the free fatty acids in an emulsion then the best way is to use a carrier, disolve the fatty acids in chlorofom and mix well with siliconized amlla glass beads, be sure to mix weel the mixture. Dry the glass beads and then add the proportion of glass beads to the BSA solution and mix continuosly at 37 degrees for 30 min. BSA will incorpórate the fatty acids. Then, in culture, add the amounto of PBS-BSA needed, previosuly filtered to setrilize the solution. If your cells are from primary culture, keep them in PBS for at least 4 hr in médium free PBS and then add the BSA-fatty acid mixture, usually activation, monitored by expression of adhesión receptors can be observed form 2 hrs.
good luck
Yes Manindra. You have to prepare a solution of bovine free fatty acid and add the fatty acid you want to incorpórate, the ratio of fatty acid per albumin is maximun 10:1, oleic acid, docosahexanoic acid or eicosapentanoic acid are easy to handle in the salt form. The solution of albumin 32 mg/ml should be in PBS 7.4 and calculate, based on the previous ratio the amount of fatty acid you have to add, mix at 37 degrees. Alternatively if you have the free fatty acids in an emulsion then the best way is to use a carrier, disolve the fatty acids in chlorofom and mix well with siliconized amlla glass beads, be sure to mix weel the mixture. Dry the glass beads and then add the proportion of glass beads to the BSA solution and mix continuosly at 37 degrees for 30 min. BSA will incorpórate the fatty acids. Then, in culture, add the amounto of PBS-BSA needed, previosuly filtered to setrilize the solution. If your cells are from primary culture, keep them in PBS for at least 4 hr in médium free PBS and then add the BSA-fatty acid mixture, usually activation, monitored by expression of adhesión receptors can be observed form 2 hrs.
good luck
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